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Experimental African Trypanosomiasis: A Subset of

Pathogenic, IFN-␥ -Producing, MHC Class II-Restricted CD4

T Cells Mediates Early Mortality in Highly Susceptible Mice1

Meiqing Shi,2 Guojian Wei,2 Wanling Pan, and Henry Tabel3

Infections of highly susceptible BALB/c mice with virulent strains of Trypanosoma congolense or Trypanosoma brucei result in

rapid death (8 days). We have previously shown that this mortality is IFN-␥ dependent. In this study we show that IFN-␥ is

produced predominantly by CD3 Thy1.2 TCR␤ CD4 T cells shortly before the death of infected mice. Mortality may therefore

be dependent on IFN-␥ -producing CD4 T cells. Surprisingly, infected CD4 / and CD4 / BALB/c mice have similar para-

sitemia and survival time. In infected CD4 / mice, the production of both IFN-␥ and IL-10 is very low, suggesting that both

cytokines are predominantly produced by CD4 T cells and that the outcome of the disease might depend on the balance of their

effects. Infected BALB/c mice partially depleted of CD4 T cells or MHC class II function have lower parasitemia and survive

significantly longer than infected normal BALB/c mice or infected BALB/c mice whose CD4 T cells are fully depleted. Partial

depletion of CD4 T cells markedly reduces IFN-␥ secretion without a major effect on the production of IL-10 and parasite-

specific IgG2a Abs. Based on our previous and current data, we conclude that a subset of a pathogenic, MHC class II-restrictedCD4 T cells (Tp cells), activated during the course of T. congolense infection, mediates early mortality in infected BALB/c mice

via excessive synthesis of IFN-␥ . IFN-␥ , in turn, exerts its pathological effect by enhancing the cytokine release syndrome of the

macrophage system activated by the phagocytosis of parasites. We speculate that IL-10-producing CD4 T cells might counteract

this effect. The Journal of Immunology, 2006, 176: 1724–1732.

African trypanosomes are single-cell parasites that infect

both humans and livestock. The parasites survive in the

bloodstream of the mammalian host and can cause se-

vere disease and death. BALB/c mice are highly susceptible to

Trypanosoma congolense and Trypanosoma brucei infections,

whereas C57BL/6 mice are relatively resistant, as measured by

levels of parasitemia, immunosuppression, and survival time (1–

3). The mechanisms of susceptibility and relative resistance inthese two mouse strains are still poorly understood. There is good

evidence that resistance/susceptibility to T. congolense infections

is controlled by at least five different quantitative trait loci on chro-

mosomes 17, 5, and 1 (4, 5). The relevant genes and their products

are unknown. The early mortality of susceptible BALB/c mice to

infection with T. congolense or T. brucei is associated with en-

hanced synthesis of IFN-␥ and IL-10 (6–8). The infection of rel-

atively resistant C57BL/6 mice is accompanied by significantly

higher plasma levels of parasite-specific IgG, in particular, IgG2a

Abs (9). The susceptible mice die of a systemic inflammatory re-

sponse syndrome (SIRS)4 (6). Administration of anti-IFN-␥ Abs to

susceptible BALB/c mice infected with T. congolense prevents

early mortality (6, 7). Blocking the IL-10R of normally relatively

resistant C57BL/6 mice infected with T. congolense leads to early

death. These mice have excessively elevated plasma levels of

IFN-␥ and monokines (6). The early mortality of C57BL/6 mice

infected with T. congolense and treated with anti-IL-10R can be

reversed by administration of anti-IFN-␥ Abs (6). Therefore, theearly mortality of these infected mice is mediated in part by IFN-␥

produced in excessive amounts (6). The fact that the early death of

infected susceptible BALB/c mice was prevented by administra-

tion of anti-IFN-␥ led us to suggest a central role for a subset of

IFN-␥ -producing cells, possibly NK cells, NKT cells, or T cells (6,

10). A novel plastic-adherent suppressor T cell population(s) in the

spleen of T. congolense-infected mice has been found to synthe-

size, in synergy with trypanosome-pulsed Thy1.2Ϫ spleen cells,

very large amounts of IFN-␥ (11). We previously speculated that

the trypanosomal GPI, the membrane anchor of the variant surface

glycoprotein (VSG), might be the major Ag involved (6). We also

speculated and started this study with the premise that the IFN-␥ -

producing cells might be CD1-restricted NKT cells (6). We now

have good evidence that the IFN-␥ -producing, pathology-inducing

T cells are not CD1 restricted (M. Shi, C.-R. Wang, G. Wei, W.

Pan, G. Appleyard, and H. Tabel, submitted for publication). The

main aim of the present study was to further characterize the subset

of IFN-␥ -producing cells that mediate a lethal process via IFN-␥ in

BALB/c mice infected with T. congolense (6). In the present study,

we use immunocytochemistry and FACS analysis to characterize

the IFN-␥ -producing T cells of BALB/c mice succumbing early to

Department of Veterinary Microbiology, Western College of Veterinary Medicine,University of Saskatchewan, Saskatoon, Canada

Received for publication August 16, 2005. Accepted for publication November10, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a research grant from the Canadian Institutes of HealthResearch (to H.T.) and a postdoctoral fellowship from the Health Services Utilizationand Research Commission of Saskatchewan (to M.S.).

2 M.S. and G.W. contributed equally to this work.

3 Address correspondence and reprint requests to Dr. Henry Tabel, Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B4. E-mail address: [email protected]

4 Abbreviations used in this paper: SIRS, systemic inflammatory response syndrome;MHC-II, MHC class II; Tp, pathogenic, MHC class II-restricted CD4ϩ T cell; Tr1,regulatory T cell; VAT, variant antigenic type; VSG, variant surface glycoprotein.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

7/28/2019 1724.full


T. congolense infection. We also test the hypothesis that the pro-

cess leading to early mortality in susceptible mice infected with a

virulent strain of T. congolense might be mediated by CD4ϩ T

cells that are restricted by the MHC class II (MHC-II).

Materials and Methods Mice

Female, 8- to 10-wk-old BALB/c AnNCrlBR (BALB/c) and 5- to 6-wk-old

female outbred Swiss white mice (CD1) were purchased from the AnimalResource Center of University of Saskatchewan. CD4 (12) knockout micewere generated in T. Mak’s laboratory (Ontario Cancer Institute and De-partment of Medical Biophysics and Immunology, University of Toronto,Toronto, Ontario, Canada). The CD4 deficiency was subsequently bred intomice with the BALB/c background in the same laboratory. The CD4Ϫ / Ϫ

BALB/c mice used in this study originated from the above colony and werebred in the animal care facilities of the Western College of VeterinaryMedicine, University of Saskatchewan. The mice were kept in polycar-bonate cages on sawdust and allowed free access to food and waterthroughout the experiments according to the recommendations of the Ca-nadian Council of Animal Care.

Parasites

T. congolense, Trans Mara strain, variant antigenic type (VAT) TC13 wasused in this study. The origin of this parasite strain has been described

previously (13). Frozen stabilates of parasites were used for infecting CD1mice immunosuppressed with cyclophosphamide, and passages were madeevery third day as described previously (13). The parasites purified fromthe blood of infected CD1 mice by DEAE-cellulose chromatography (14)were used for infecting BALB/c mice.

Hybridomas and Abs

The rat hybridomas GK1.5 (specific for CD4) and HB3 (specific for I-Ad)were purchased from American Type Culture Collection. The followingmAbs were purchased from BD Pharmingen: purified anti-mouse CD16/ CD32 (Fc␥ III/IIR, clone 2.4G2), purified rat anti-mouse CD3 (clone17A2), biotin-conjugated rat anti-mouse CD4 (RM4-5), biotin-conjugatedrat anti-mouse CD8 (53-6.7), biotin-conjugated hamster anti-mouse TCR␤(H57-597), biotin-conjugated anti-mouse Pan-NK cells (DX5), biotin-con-

jugated rat anti-mouse IFN-␥ (XMG1.2), and FITC-conjugated rat anti-mouse IFN-␥ (XMG1.2). The following Abs were purchased from Cedar-

lane Laboratories: biotin-conjugated goat anti-rat IgG (HϩL) mouseadsorbed, biotin-conjugated anti-mouse Thy1.2 (5a-8), biotin-conjugatedrat anti-mouse CD11b (M1/70.15), biotin-conjugated hamster anti-mouseTCR␥␦ (GL3), purified rat IgG2b, biotin-conjugated rat IgG2a, biotin-con-

jugated rat IgG2b, biotin-conjugated rat IgG1, biotin-conjugated rat IgM,biotin-conjugated hamster IgG, and FITC-conjugated rat IgG1.

Infections, treatment of mice with mAbs, estimation of

parasitemia, and survival time of mice

Mice were infected i.p. with 103 T. congolense VAT TC13. Some groupsof infected BALB/c mice were i.v. injected with a single dose of 4 mg, 500g, 100 g, or 30 g of anti-CD4 mAb on day 0 after infection. Othergroups of infected mice were injected with 400 g of anti-I-Ad on days 0,3, 5, and 7. A drop of blood was taken from the tail of each infected mouse.Parasitemia was estimated by counting the number of parasites present in

at least 10 fields atϫ

400 magnification by phase contrast microscopy. Thesurvival time was defined as the number of days after infection that theinfected mouse remained alive.

Immunocytochemistry

Spleen cells were isolated from T. congolense-infected BALB/c mice onday 7 after infection or from uninfected BALB/c mice (as control). Thecells (107 /ml) in complete medium were seeded into tissue culture-treatedplastic petri dishes (Falcon 3001; VWR International, Edmonton Alberta,Canada) and incubated at 37°C in a 5% CO2 incubator. After 3 h, thenonadherent cells were removed. The adherent cells were carefully washedand then dislodged with ice-cold versene (0.02% EDTA in PBS (pH 7.2))and gentle scraping. The cells were diluted at 2.5 ϫ 106 cells/ml. Fourhundred and twenty-five microliters of the cell solution was put in eachchamber of eight-chamber Lab-Tek slides (Miles Scientific) and culturedfor 48 h at 37°C in a 5% CO

2atmosphere. Then the chamber slides were

disassembled. Immunofluorescent double staining for cell surface markersand intracellular IFN-␥ was performed on ice. In brief, the cells were rinsedwith PBS, blocked with Fc block (purified anti-mouse CD16/CD32

(Fc␥ III/IIR), clone 2.4G2) and 2% BSA, then incubated with biotin-conjugated anti-mouse Thy1.2, CD3, CD4, CD8, TCR␤, TCR␥␦, CD11b, orpan NK cell mAb or biotin isotype controls for 30 min. After three washesin PBS, the slides were incubated with streptavidin AlexaFluor 488 (Mo-lecular Probes) for 30 min, rinsed with PBS, then fixed with 5% formalin.The cells were incubated with avidin D solution for 15 min, rinsed withPBS, then incubated with biotin solution (avidin/biotin blocking kit; VectorLaboratories) for 15 min. The slides were rinsed with PBS, then incubatedwith biotinylated rat anti-mouse IFN-␥ (XMG1.2) or rat IgG1 conjugatedwith biotin (isotype control) in PBS containing 0.1% saponin (cell perme-

abilization) for 30 min, rinsed with PBS, then stained with streptavidinAlexaFluor 546.

Flow cytometry

Spleen cells were collected from T. congolense-infected BALB/c mice onday 7 after infection or from uninfected BALB/c mice (as a negative con-trol). The cells were diluted to 5 ϫ 106 cells/ml and cultured (200 l/well)in a 96-well plate for 48 h. Spleen cells from uninfected mice were stim-ulated with 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma-Aldrich) as a positive control. After 44 h of incubation, 2 M monensin(GolgiStop; BD Pharmingen) was added to the cultures. Four hours later,cells were harvested and washed twice in staining buffer (BD Pharmingen).The cells were incubated (15 min, 4°C) with purified mAb 2.4G2 to block nonspecific binding of Abs to FcRs, washed with staining buffer, resus-pended in staining buffer, and surface stained with the relevant biotinylatedAbs. Cells were washed with staining buffer, labeled with PE-conjugated

strepavidin (BD Pharmingen), and washed twice. Intracellular staining wasperformed using the Cytofix/CytoPerm kit (BD Pharmingen) in accordancewith the manufacturer’s recommendations. Briefly, cells were treated withformaldehyde and saponin to fix and permeabilize the cells. Intracellularstaining was then performed using FITC-conjugated anti-IFN-␥ mAb(XMG1.2) and FITC-conjugated rat IgG1 (isotype-matched control Ab).Samples were resuspended in PBS containing 1% formaldehyde, tested byFACS, and analyzed using CellQuest software (BD Biosciences).

Splenocyte cultures for measurement of cytokine synthesis

Splenocytes were collected from T. congolense-infected CD4Ϫ / Ϫ or wild-type of BALB/c on day 7 after infection as well as from relevant uninfectedmice. Cells were cultured at a concentration of 5 ϫ 106 cells/ml (200l/well) in 96-well tissue culture plates in a humidified incubator contain-ing 5% CO

2in the air. The culture supernatant fluids were collected after

48 h and centrifuged at 1500 ϫ g for 10 min, and the supernatant fluids

were stored for cytokine assays at Ϫ35°C until used.

Cytokine assays

Recombinant murine cytokines (IFN-␥ , IL-10, and IL-12p40) and Abs tothese cytokines for use in ELISA were purchased from BD Pharmingen orR&D Systems. The levels of cytokines in culture supernatant fluids orplasma were determined by routine sandwich ELISA using Immuno-4plates (Dynax Technologies) according to the manufacturer’s protocols.Each sample was tested for each cytokine in triplicate.

ELISA for trypanosome-specific Abs

Whole trypanosome lysate was prepared by three cycles of freezing andthawing of freshly isolated T. congolense VAT TC14 in the presence of 5mM N -tosyl-L-lysine chloromethylketone (Sigma-Aldrich), and the totalprotein content was determined using a protein assay kit (Bio-Rad). ELISA

plates were coated overnight at 4°C with 50 l of the lysate containing 25g/ml total protein. The plates were washed twice with PBS/Tween 20,and nonspecific binding sites were blocked for 2 h at room temperaturewith 200 l of PBS containing 10% heat-inactivated FBS (PBS-FBS).Serum samples (100 l), diluted 1/50 in PBS-FBS were added to each welland incubated for 2 h at 37°C. After washing (four times), 100 l of previously determined dilutions of peroxidase-conjugated goat anti-mouseisotype-specific Abs (Southern Biotechnology Associates) in PBS-FBSwere added to each well and incubated for 2 h at room temperature. Theplates were washed eight times, and color development was achieved byadding 100 l of ABTS (Kirkegaard & Perry) and incubating for 15–30min at room temperature. ODs were read in a microtiter plate reader at awavelength of 405 mm.

Statistical analysis

Data are represented as the mean Ϯ SE. Significance of differences was

determined by ANOVA using StatView SE 1988 software (Abacus Con-cepts) or a log-rank test for curve comparison using a PRISM computerprogram (GraphPad).

1725The Journal of Immunology

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Results IFN-␥ is produced predominantly by

CD3ϩThy1.2ϩTCR␤ϩCD4ϩ T cells in BALB/c mice infected

with T. congolense

To characterize the IFN-␥ -producing cells, immunocytochemical

double staining for cell surface markers and intracellular IFN-␥

was performed. All cell cultures stained with control Abs of the

same isotype were negative. We found IFN-␥ -producing cells in

spleen cultures of mice infected with T. congolense. In contrast, noIFN-␥ -positive cells were detected in spleen cultures of uninfected

mice. The IFN-␥ -producing cells were glass adherent and fre-

quently located close to large cells that had the morphology of

macrophages or dendritic cells (see insets of Fig. 1 A). The IFN-

␥ -producing cells were DX5Ϫ (Fig. 1 B) and CD11bϪ (Fig. 1 D).

This indicated that they were neither NK cells nor macrophages. All

IFN-␥ -positive cells expressed CD3 (Fig. 1 A) and Thy1.2 (Fig. 1C )

marker. Thus, the IFN-␥ -secreting cells were T cells. We also found

that IFN-␥ -producing cells were TCR␤ϩ (Fig. 1 E ) and ␥␦Ϫ (Fig. 1F ).

The vast majority (90%) of the IFN-␥ -producing cells were CD4ϩ

(Fig. 1G). However, some IFN-␥ -producing cells were CD4Ϫ (Fig.

1G). Four percent of IFN-␥ -producing cells were CD8ϩ (Fig. 1 H ).

Thus, the immunocytochemical analysis showed that IFN-␥ was pre-

dominantly produced by CD3ϩ

Thy1.2ϩ

TCR␤ϩ

CD4ϩ

cells. When incontact with a CD11bϩ cell, IFN-␥ -producing T cells were polarized,

i.e., the intracellular IFN-␥ was concentrated at the area of cell-cell

contact (Fig. 1 D).

Next, we checked the IFN-␥ -producing cells using double stain-

ing and flow cytometry. No positive cells were found when cells

FIGURE 1. Characterization of the

IFN-␥ -producing cells in spleen cul-

tures of mice infected with T. congo-

lense by immunofluorescent double

staining. BALB/c mice were infected

with 103 T. congolense. Plastic-adher-

ent spleen cells were collected, as de-

scribed in Materials and Methods,

from infected mice on day 7 after in-

fection and cultured at concentration of

2.5 ϫ 106

cells/ml in chamber slides.Immunofluorescent double staining for

cell surface phenotype and intracellular

IFN-␥ was performed as described in

Materials and Methods. Original mag-

nification, ϫ400; for insets, ϫ1000.

IFN-␥ -producing cells (red, arrows)

were CD3ϩ (green; A) and DX5Ϫ

(green; B): Note that the intracellular

IFN-␥ is polarized toward the neigh-

boring cell. C , Thy1.2ϩ (green); D,

CD11bϪ (green). Note that the IFN-␥

within the T cell is polarized toward

the area of contact with the CD11bϩ

cell. E , TCR␤ϩ (green); F , TCR␥␦Ϫ

(green). G, The vast majority of IFN-␥ -producing cells (90%) (red, arrows)

had the CD4 marker (green), although

some IFN-␥ ϩ CD4Ϫ cells were de-

tected. H , A small portion of IFN-␥ -

producing cells was CD8ϩ (4%;

green).

1726 CD4ϩ T CELLS AND EARLY MORTALITY IN T. congolense INFECTIONS

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from mice infected with T. congolense were stained only with

isotype control Ab (Fig. 2). The IFN-␥ -producing cells expressed

Thy1.2, TCR␤, and CD4 markers (Fig. 2). No IFN-␥ -secreting

cells were detected in spleen cultures of uninfected mice (data not

shown). Thus, FACS data confirmed that the IFN-␥ -producing

cells were TCR␤ϩCD4ϩ T cells and a small subset (0.5%) of the

total number of spleen cells (Fig. 2).

Parasitemia and survival time of CD4Ϫ / Ϫ BALB/c mice infected

with T. congolense are not different from those of infected wild-

type BALB/c mice

We formulated the hypothesis that CD4Ϫ / Ϫ BALB/c mice infected

with T. congolense would have lower parasitemia and survive

longer than infected wild-type BALB/c mice, because IFN-␥ was

predominantly produced by CD4ϩ T cells, and we had demon-

strated that IFN-␥ mediated early mortality (7, 11). Examination of

CD4Ϫ / Ϫ and wild-type BALB/c mice after infection with T. con-

golense showed that there were no significant differences in either

parasitemia (Fig. 3 A) or survival time (Fig. 3 B). Thus, our obser-

vations did not support our hypothesis. We concluded that infec-

tions of CD4 knockout mice are an experimental design of limited

usefulness and might yield misleading results. One has to keep in

mind that CD4 molecules are expressed on a number of subsets of

T cells with different, if not opposing, functions. These CD4ϩ T

cells would include IFN-␥ -producing Th1 cells (15), IL-10-pro-

ducing regulatory T cells (Tr1) (16), and, as postulated in this

study, IFN-␥ -producing pathogenic T cells. This led us to explore

the possibility that the IFN-␥ , shown to be critical for mediating

death, was produced by a special subset of CD4ϩ T cells.

Synthesis of IFN-␥ and IL-10 is markedly lower in spleen

cultures from CD4Ϫ / Ϫ BALB/c mice infected with T. congolense

than in spleen cultures from infected wild-type BALB/c mice

We assessed whether spleen cells from infected CD4Ϫ / Ϫ BALB/c

mice secreted IFN-␥ and IL-10. We measured the levels of IFN-␥

as well as IL-10 in spleen cell cultures from CD4Ϫ / Ϫ and CD4ϩ / ϩ

BALB/c mice infected with T. congolense. Besides IFN-␥ , IL-10

is also significantly enhanced in susceptible BALB/c mice infected

with T. congolense (8) and is crucial for controlling the cytokinerelease syndrome and the development of SIRS (6). Spleen cul-

tures from infected mice always contain a certain number of par-

asites and, hence, parasite Ag. Supernatant fluids of spleen cell

cultures derived from infected CD4ϩ / ϩ and CD4Ϫ / ϪBALB/c mice

had 583 pg of IFN-␥ /ml and 17 pg of IFN-␥ /ml, respectively (Fig.

3C ). Thus, the spleen cell cultures of infected CD4Ϫ / Ϫ mice only

yieldedϳ3% the IFN-␥ produced by the cultures of infected wild-

type mice ( p Ͻ 0.01). This small amount of IFN-␥ was possibly

produced by CD8ϩ T cells (Fig. 1 H ). A major involvement of

IFN-␥ -producing CD8ϩ T cells in the IFN-␥ -mediated death ap-

peared unlikely, because CD8-deficient BALB/c mice do not sur-

vive longer after infection than wild-type BALB/c mice (data not

shown). Again, these data support the already drawn conclusion

that IFN-␥ is predominantly produced by a small subset of CD4ϩ

T cells in infected wild-type BALB/c mice.

Our observations had led us to conclude that the T cells centrally

involved in causing early death in infected BALB/c mice are most

probably CD4ϩ T cells as described above (Fig. 1). Our observa-

tions also show that the parasitemia and survival time are similar

in CD4Ϫ / Ϫ and CD4ϩ / ϩ mice after infection (Fig. 3). To try to

resolve this paradox, we measured the production of IL-10, be-

cause IL-10 is crucial in preventing SIRS in T. congolense-infected

mice (6). The spleen cell cultures of infected CD4Ϫ / Ϫ BALB/c

mice produced 4.5-fold less IL-10 ( p Ͻ 0.01) than those from

infected wild-type BALB/c mice, amounts similar to those pro-

duced by uninfected spleen cell cultures (Fig. 3 D). Thus, our data

demonstrate that the synthesis of IL-10 in T. congolense-infected

BALB/c mice is CD4ϩ T cell dependent.

FIGURE 2. IFN-␥ -producing cells in cell cultures of whole spleen from

mice infected with T. congolense: characterization by flow cytometry.

Groups of six BALB/c mice were infected with 103 T. congolense. Spleen

cells collected on day 7 after infection from infected mice were cultured at

concentration of 5 ϫ 106 cells/ml in 96-well plates (200 l/well) for 48 h,

and 2 M monensin (GolgiStop; BD Pharmingen) was added to the culture

at 44 h. Double staining for cell surface phenotype and intracellular IFN-␥

was performed as described in Materials and Methods. IFN-␥ -producing

cells are Thy1.2

ϩ

( D

; isotype control,A

), TCR␤

ϩ

( E

; isotype control,B

),and CD4ϩ (F ; isotype control, C ). The results presented are representative

of two separate experiments.

FIGURE 3. In CD4Ϫ / Ϫ BALB/c mice infected with T. congolense, par-

asitemia and survival were not significantly different, but secretion of

IFN-␥ and IL-10 was decreased by Ͼ97%. Groups of 10 CD4Ϫ / Ϫ (E) or

wild-type BALB/c (F) mice were infected with 103 TC13. Mice were

monitored for parasitemia ( A) and survival ( B). Groups of four CD4Ϫ / Ϫ

(Ⅺ) or wild-type (f) BALB/c mice were infected with 103 TC13. Spleen

cells collected on day 7 after infection from infected mice were cultured at

concentration of 5 ϫ 106 cells/ml in 96-well plates (200 l/well) for 48 h.

The culture supernatant fluids were assayed for IFN-␥ and IL-10, as de-

scribed in Materials and Methods. Data are presented as the mean Ϯ SE.

The results presented are representative of two separate experiments.


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Production of parasite-specific IgG2a, but not IgM, Abs is lower

in infected CD4Ϫ / Ϫ BALB/c than in infected wild-type mice

We measured the plasma levels of Abs specific for T. congolense,

because anti-VSG Abs play a significant role in the clearance of

the parasitemia (17–19), and Abs to common Ags also mediate a

protective role in African trypanosomiasis (20, 21). We did not

find any differences in plasma levels of IgM Abs specific for T.

congolense (Fig. 4 A) in CD4Ϫ / Ϫ and wild-type BALB/c mice in-

fected with T. congolense. However, IgG2a Abs (Fig. 4C ) to T.congolense were abrogated, and total parasite-specific IgG Abs

(Fig. 4 B) were significantly reduced in CD4Ϫ / Ϫ mice compared

with infected wild-type mice ( p Ͻ 0.01). Thus, infected CD4Ϫ / Ϫ

mice are expected not only to lack the subset of IFN-␥ -producing

CD4ϩ T cells mediating early death, but also a subset of parasite-

specific CD4ϩ Th cells that produce IFN-␥ and support the switch

from production of IgM Abs to parasite-specific IgG2a Abs.

The high plasma levels of IL12p40 do not differ in CD4Ϫ / Ϫ and

CD4ϩ / ϩ BALB/c mice infected with T. congolense

Both CD4Ϫ / Ϫ and CD4ϩ / ϩ BALB/c mice infected with T. congo-

lense succumbed to the infection without control of the first par-

asitemia (Fig. 3, A and B). Infected CD4Ϫ / Ϫ BALB/c mice pro-

duced neither IFN-␥ nor significant amounts of IL-10 (Fig. 3, C

and D). We asked ourselves: why did the CD4Ϫ / Ϫ mice die in the

absence of IFN-␥ ? One can make the following arguments: 1) The

primary excessive activation of the macrophage system happens

by phagocytosis of trypanosomes mediated by anti-VSG Abs of

IgM class. 2) Whether the development of SIRS and death occurs

does not only depend on the superimposed effect of IFN-␥ , but also

the counterbalancing effect of IL-10 that down-regulates macro-

phage activity (22). In fact, we and other investigators (6, 23) have

shown the absolute requirement for IL-10 activity to prevent try-

panosome-induced death. If the above reasoning is correct, plasma

levels of monokines should be high in infected CD4Ϫ / Ϫ BALB/c

mice that lack significant amounts of IFN-␥ and IL-10. Thus, we

measured the plasma levels of the monokine IL-12p40 in infectedCD4Ϫ / Ϫ BALB/c mice. The result showed that plasma levels of

IL-12p40 are, indeed, very high (Fig. 5).

Anti-CD4 mAb administered to infected BALB/c mice decreases

parasitemia and significantly enhances survival time when given

in optimal doses, but not when given in high doses

It seemed to us that more complex mechanisms than we initially

had anticipated must determine the outcome of T. congolense in-

fections of CD4Ϫ / Ϫ BALB/c mice. We thought that administration

of a certain optimal amount of anti-CD4 mAb might remove suf-

ficient numbers of the IFN-␥ -producing, death-inducing T cells

and hence might reduce the production of IFN-␥ below a pathol-

ogy-inducing level. Such treatment, of course, could only work if it did not detrimentally affect IL-10-producing T cells and did not

affect the protective T cells that may act by helping the generation

of protective antiparasite-specific IgG2a Abs. We therefore treated

groups of T. congolense-infected CD4ϩ / ϩ BALB/c mice with 4

mg, 500 g, 100 g, or 30 g of anti-CD4 mAb (GK1.5) on day

0. The injection of 30 g of anti-CD4 mAb had no effect (not

shown). The infected mice treated with the high dose (4 mg) of

anti-CD4 showed a pattern of parasitemia (Fig. 6 A) and survival

(Fig. 6 B) similar to that of infected CD4Ϫ / Ϫ BALB/c mice (Fig. 3,

A and B). FACS analysis demonstrated that Ͼ99% of CD4ϩ T

cells in these mice were eliminated (not shown). The groups of

infected mice treated with 500 or 100 g of anti-CD4 mAb, how-

ever, had lower parasitemia (Fig. 6 A) and significantly longer sur-vival time (Fig. 6 B) than the untreated infected BALB/c mice. As

determined by FACS, infected BALB/c mice injected with 100 g

of anti-CD4 mAb had 60% less CD4ϩ T cells than untreated in-

fected mice on day 6. In addition, these T cells expressed, on the

average,ϳ10-fold fewer CD4 molecules per cell than T cells from

untreated mice (Fig. 6C ).

Partial depletion of CD4ϩ T cells in vivo significantly reduces

IFN-␥ secretion in spleen cell cultures without strongly affecting

secretion of IL-10 and plasma levels of IgG2a Abs specific for

T. congolense

Next, we assessed the IFN-␥ and IL-10 secretion in spleen cultures

and plasma levels of IgG2a Abs specific for T. congolense on day7 after infection after treatment with anti-CD4 mAb. As shown in

Fig. 7 A, IFN-␥ secretion was significantly reduced by treatment

with a low dose (0.1 mg) of mAb and was almost abrogated by

treatment with a high dose (4 mg) of mAb. In contrast, there was

no significant difference between untreated mice and mice treated

with an optimal dose (0.1 mg) of mAb regarding the plasma levels

of IgG2a Abs specific for T. congolense. The plasma levels of

IgG2a Abs specific for T. congolense, however, were significantly

reduced by treatment with a high dose (4 mg) of anti-CD4 mAb

(Fig. 7 B). After treatment with the low dose of anti-CD4 (0.1 mg),

secretion of IL-10 by spleen cell cultures was not strongly reduced

(Fig. 7C ). Taken together, these results suggest that removal of a

certain number of pathogenic CD4ϩ T cells by treatment with an

optimal amount of anti-CD4 enhances the survival of the suscep-

tible mice infected with T. congolense. Such treatment prevents the

FIGURE 4. Synthesis of IgG2a (but not IgM) parasite-specific Abs was

abrogated in CD4Ϫ / ϪBALB/c mice infected with T. congolense. Groups of

four CD4Ϫ / Ϫ (E) or wild-type (F) BALB/c mice were infected with 103

TC13. Plasma samples were collected from the infected mice on days 0, 6,

and 7 after infection. The Ab levels in plasma were measured by ELISA asdescribed in Materials and Methods. Data are presented as the mean Ϯ SE.

The results presented are representative of two separate experiments.

FIGURE 5. Plasma levels of IL-12p40 in CD4Ϫ / Ϫ BALB/c mice in-

fected with T. congolense are as high as those in infected CD4ϩ / ϩBALB/c

mice. Groups of five mice were infected with 103 TC13, and blood samples

were collected 7 days after infection. Concentrations of IL-12p40 in the

plasma were measured by ELISA as described in Materials and Methods.

Data are presented as the mean Ϯ SE. The results presented are represen-

tative of two separate experiments.


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excessive synthesis of IFN-␥ . At the same time, this treatment

allows the production of sufficient down-regulatory IL-10 as well

as synthesis of sufficient parasite-specific IgG2a Abs.

Partial blocking of MHC-II leads to control of the first

parasitemic wave, increases the survival, and markedly reduces

the production of IFN-␥ , but not IL-10, of highly susceptible

BALB/c mice infected with T. congolense

To test whether the pathogenic, IFN-␥ -producing T cells might be

dependent on MHC-II, we treated infected BALB/c mice with anti-I-Ad mAbs (24). Whereas the infected control mice died with ful-

minating parasitemia (Fig. 8 A), the majority of infected mice

treated with anti-I-Ad mAbs controlled the first wave of para-

sitemia (Fig. 8 A). The survival time of the infected mice treated

with anti-I-Ad mAbs was significantly enhanced (Fig. 8 B). In the

spleen cultures of infected mice treated with anti-I-Ad mAbs, when

tested on day 7, the production of IFN-␥ was reduced by Ͼ10-fold

(Fig. 8C ), and the synthesis of IL-12p40 was reduced by 50% (not

shown), compared with infected control mice. The production of

IL-10 by the spleen cell cultures did not seem to be altered very

much by anti-I-Ad mAb treatment of the infected mice (Fig. 8 D).

DiscussionWe have provided evidence that highly susceptible BALB/c

mice infected with T. congolense die of SIRS that is mediated

by IFN-␥ (6, 25). This SIRS is associated with elevated plasma

levels of IL-6, IL-12p40, IL-10, and IFN-␥ ; focal liver lesions

of apoptotic parenchymal cells; 5-fold enlargement of Kupffer

cells; apoptosis of 10% of Kupffer cells; enlarged capillary bed;

hypotension; decreased body temperature; piloerection; and hy-

pomotility. These observations prompted us to characterize the

IFN-␥ -producing, pathogenic cells. Uzonna et al. (11) discov-

ered a novel plastic-adherent subset of suppressor T cells in

spleens of T. congolense-infected BALB/c mice. These T cells

were characterized as CD3ϩThy1.2ϩ and either CD4ϩ or

CD4Ϫ8Ϫ by Ab-mediated depletion. This subset of T cells was

found to exert its immunosuppressive effect via IFN-␥ (11). We

have shown that this subset of T cells is not CD1 restricted. By

both immunocytochemistry and FACS, we have provided direct

evi dence t hat m ost of the I FN-␥ -producing cells are

CD3ϩThy1.2ϩTCR␤ϩCD4ϩ in wild-type BALB/c mice in-

fected with T. congolense (Figs. 1 and 2). In contrast to wild-

type BALB/c mice, very little IFN-␥ was detected in the spleen

cultures of CD4Ϫ / Ϫ BALB/c mice infected with T. congolense

(Fig. 3C ). This indicates that most of the IFN-␥ is produced by

CD4ϩ T cells.

Because we found that CD4ϩ T cells produced most of the

IFN-␥ that mediated early mortality during the course of infectionwith T. congolense, we anticipated that CD4Ϫ / Ϫ BALB/c mice

infected with T. congolense would survive longer than the infected

wild-type BALB/c mice. To our surprise, the total deficiency of

CD4ϩ T cells did not alter the parasitemia and survival time after

infection (Fig. 3, A and B). Why would infected CD4Ϫ / Ϫ BALB/c

mice still die when IFN-␥ -producing CD4ϩ T cells were absent? It

appeared to us that the operating mechanisms were more complex

than we had initially anticipated. We concluded that infections of

CD4 knockout mice are an experimental design of limited useful-

ness for this study. Our reasoning was based on the observation

that CD4 molecules are expressed on a number of subsets of T

cells with different, if not opposing, functions. The population of

CD4

ϩ

T cells include subsets of IFN-␥ -producing Th1 cells (15),IL-10-producing Tr1 (16), and, as postulated in this study, IFN-

␥ -producing pathogenic T cells. It is plausible that the excessive

amount of IFN-␥ is only one of many pathogenic factors. The

pathogenic effect of IFN-␥ can be viewed as an amplifier of the

cytokine release syndrome of trypanosome-pulsed macrophages

that we reported and discussed previously (6). We found that

CD4Ϫ / Ϫ mice infected with T. congolense produced significantly

less total parasite-specific IgG Abs and produced almost no para-

site-specific IgG2a Abs compared with infected wild-type mice

(Fig. 4). The levels of parasite-specific IgM Abs were not affected.

These results support the idea that IgM production is predomi-

nantly CD4ϩ T cell independent (26–28). Presumably, the switch

from IgM to IgG (in particular, IgG2a) requires help from CD4ϩ

Th1 cells. We suggest that in T. congolense infections of CD4Ϫ / Ϫ

BALB/c mice, the beneficial effect of a substantial decrease in

FIGURE 6. Low-dose, but not high-dose, anti-CD4

treatment enhances the survival of highly susceptible

BALB/c mice infected with T. congolense. Groups of five

BALB/c mice were infected with 103 TC13 and treated

with 4 mg (Ⅺ), 0.5 mg (‚), or 0.1 mg (E) of anti-CD4 or

were left untreated (F) as a control on day 0 after infec-

tion, respectively. Mice were monitored for parasitemia

( A) and survival ( B). FACS analysis was performed for

CD4ϩ spleen cells of infected mice untreated or treated

with 0.1 mg/ml anti-CD4 Abs (C ). The results presented

are representative of two separate experiments.


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IFN-␥ is partially abrogated by 1) lack of the protective parasite-

specific IgG Abs, and 2) lack of down-regulatory IL-10.

The development of IgG Abs to trypanosomal cysteine protein-

ase is associated with resistance of cattle to the disease caused by

T. congolense infections (20, 21). For the time being, the above

explanation is incomplete, because presently we do not know the

nature of the murine IgG anti-parasite Abs measured by ELISA.

We have evidence that antiparasite Abs of IgG3 and IgG2a isotype

are correlated with resistance to T. congolense infections of mice

(9). In a study of IgG2a vs IgM Abs to VSG of T. congolense, we

found that both types of Abs were equally effective in mediating

phagocytosis of T. congolense by macrophages. IgG2a anti-VSG-mediated phagocytosis, however, induces the production of more

NO by trypanosome-pulsed macrophages than IgM anti-

VSG-mediated phagocytosis (29). NO produced by macrophages

is known to be cytotoxic to T. brucei (30) and T. congolense (29).

T. congolense-infected CD4Ϫ / Ϫ BALB/c mice lacked IL-10

production (Fig. 3), indicating that most of the elevated IL-10 pro-

duction in infected mice is dependent on CD4ϩ T cells. We sug-

gest that T. congolense-infected CD4Ϫ / Ϫ BALB/c mice might lack

Tr1 producing IL-10. IL-10-producing Tr1 have been shown to

play a role in infections of Bordetella pertussis (16, 31) and Leish-

mania major (32). We and others (6, 23) have shown that IL-10

function is absolutely required to prevent early death in trypano-

some-infected, relatively resistant mice. IL-10 predominantly ex-erts its down-regulatory activity on T cells by down-regulating

APCs (22), such as macrophages. The lack of high levels of IL-10

(Fig. 3) might explain the presence of highly activated macro-

phages, as shown by high plasma levels of monokines (Fig. 5) and

the development of SIRS in infected CD4Ϫ / Ϫ BALB/c mice (Fig.

3) despite the lack of activity of Tp cells (Fig. 3).

The unexpected results of infection of CD4Ϫ / Ϫ BALB/c mice

prompted us to investigate whether partial depletion of CD4ϩ T

cells might decrease the secretion of IFN-␥ without significantly

reducing the production of IgG Abs and IL-10. We reasoned that

an optimal dose of anti-CD4 might sufficiently affect the small, but

powerful, population of Tp cells without strongly affecting the

other subsets of CD4ϩ T cells. To this end, we treated infected

wild-type BALB/c mice with varying doses of anti-CD4 Abs. The

administration of 4 mg of anti-CD4 Abs to infected BALB/c mice

eliminated Ͼ99% of the CD4ϩ

T cells and thus was similar to thesituation in CD4Ϫ / Ϫ mice. It did not significantly alter parasitemia

or survival time, compared with untreated infected controls (Fig.

6). However, the administration of 0.5 mg, and, even more so, 0.1

mg of anti-CD4 mAb resulted in decreased parasitemia and en-

hanced survival time (Fig. 6). These results suggest that removal of

a certain number of CD4ϩ Tp cells from T. congolense-infected

BALB/c mice by treatment with an optimal amount of anti-CD4

enhances the survival of the infected mice. Such treatment pre-

vents the excessive synthesis of IFN-␥ (Fig. 7 A). At the same time,

this treatment allows the production of sufficient down-regulatory

IL-10 (Fig. 7 B) as well as the synthesis of sufficient parasite-spe-

cific IgG2a Abs (Fig. 7C ). The administration of an optimal

amount of anti-I-A

d

mAbs to T. congolense-infected BALB/c micehad a similar effect as the anti-CD4 treatment. The treated mice

controlled the first parasitemic wave (Fig. 8 A), had longer survival

time (Fig. 8 B), and produced significantly less IFN-␥ (Fig. 8C )

without affecting the synthesis of IL-10 (Fig. 8 D). These results

strongly suggest that the IFN-␥ -producing Tp cells are MHC class

II restricted. This conclusion is supported by the observation that

early IFN-␥ -mediated death occurs in infected relatively resistant

C57BL/6 mice after treatment with anti-IL10R Abs (6), but not in

infected MHC-II-deficient C57BL/6 mice treated equally (M. Shi,

G. Wei, and H. Tabel, unpublished observations).

Why did the administration of anti-CD4 or anti-MHC-II pref-

erentially abolish the function of CD4ϩ Tp cells? We presently do

not know; we can only speculate. We do know from our FACS

analysis that the administration of 0.1 mg of anti-CD4 reduced the

total CD4ϩ T cell population of the spleen by ϳ60% and the

FIGURE 7. Partial depletion of CD4ϩ T cells in T. congolense-infected

BALB/c mice markedly reduces IFN-␥ secretion in spleen cell cultures

without a strong effect on IL-10 secretion and plasma levels of parasite-

specific IgG2a Abs. Groups of five BALB/c mice were infected with 103

TC13 and treated with 4 or 0.1 mg of anti-CD4 or were left untreated on

day 0. On day 7 after infection, spleen cells were collected from infected

mice and cultured (5 ϫ 106 cells/ml) in 96-well plates (200 l/well) for

48 h. The culture supernatant fluids were assayed for IFN-␥ and IL-10 as

described in Materials and Methods. Plasma levels of IgG2a Abs specific

for T. congolense were also assessed as described in Materials and Meth-

ods. Data are presented as the mean Ϯ SE. The results presented are rep-

resentative of two separate experiments.

FIGURE 8. Partial blocking of MHC-II leads to increased survival of

highly susceptible BALB/c mice infected with T. congolense, associated

with control of the first parasitemic wave and reduced production of IFN-␥

without significantly affecting IL-10 production. Groups of five BALB/c

mice were infected with 103 TC13 and treated with 400 g of Ab specific

for I-Ad or rat IgG on days 0, 3, 5, and 7 after infection and compared with

infected mice that did not receive Ab treatment. Mice were monitored for

parasitemia ( A) and survival ( B). Spleen cell cultures were performed as

described in Materials and Methods. The concentrations of IFN-␥ (C ) and

IL-10 ( D) in the supernatant fluids were determined by ELISA. Data are

presented as the mean Ϯ SE. The results presented are representative of

two separate experiments.


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average expression of CD4 molecules per cell by ϳ90% (Fig. 6C ).

We speculate that the TCRs of CD4ϩ Tp cells might have a low

affinity for the parasite Ag(s). Thus, Tp cells might require the

binding of more clusters of TCR/CD4/MHC complexes to be ac-

tivated than the other subsets of CD4ϩ T cells require.

We discuss our data with the use of a hypothetical simplistic

diagram. We are aware that the presented synopsis is incomplete,

but we suggest that it is coherent and compatible with presently

known facts (Fig. 9). 1) IgM anti-VSG, initially produced inde-

pendently of T cells (26–28), binds to the circulating trypano-

somes, and 2) mediate phagocytosis of the opsonized parasites (19,

33). 3) The trypanosome-pulsed macrophages produce monokines

(19, 33, 34). 4) The pulsed macrophages process the parasites and

present parasite Ag via surface MHC class II to CD4ϩ T cells. 5)

A subset of activated pathogenic CD4ϩ T cells (Tp cells) produce

IFN-␥ , which, in turn, further activates the macrophages and in-

duces the macrophages to produce enhanced amounts of mono-

kines, such as TNF-␣, IL-1, IL-6, IL-12, and NO, etc. (19, 29, 34).

6) The macrophages also produce IL-10 (34), which, via an auto-

crine pathway, down-regulates macrophages in a negative feed-

back loop. 7) Presently, we have no direct evidence for the in-

volvement of regulatory T cells. However, because we and others

have provided evidence that IL-10 is absolutely necessary for pre-venting early death in trypanosome-infected, relatively resistant

mice (6, 23), and the production of the high amounts of IL-10 in

T. congolense infections is dependent on CD4ϩ T cells (Fig. 3 D),

we speculate that some of the IL-10 might be produced by Tr1.

Ag-specific CD4ϩ Tr1 might produce IL-10 down-regulating

CD4ϩ Tp cells as well as macrophages (6). When activation of

macrophages, predominantly Kupffer cells of the liver (6, 19), is

exceeding a certain threshold, excessive SIRS will lead to death.

Thus, the state of macrophage activation (25) is determined by at

least three major factors: phagocytosis of trypanosomes, enhancing

effect of IFN-␥ , and down-regulatory effect of IL-10 (6).

The main aim of this study was to shed light on the cell(s) that

produces the excessive amounts of IFN-␥ that, in turn, mediateearly mortality of mice infected with T. congolense. Although we

have provided evidence that the mortality-inducing cell is a subset

of MHC-II-restricted CD4ϩ T cells, many questions remain unan-

swered. For what Ag is the Tp cell specific? Are there many par-

asite Ags involved or one major Ag only? Although we have no

direct evidence, we consider the glycosylinositolphosphate (35,

36) of the GPI anchor of the VSG as a possible candidate. GPI of

T. brucei is a virulence factor of its own, by inducing macrophages

to produce monokines (36, 37). Is the subset of CD4ϩ Tp cells

simply a hyperactive Th1 cell or is it a subset with unique surface

markers? The pathogenic T cells described in this study have all

the properties of Th1 cells; however, their matrix adherence (ad-

herent to plastic, glass, and nylon wool) (11) distinguishes this

subpopulation of T cells from conventionally called Th1 cells.

How is the IFN-␥ -producing Tp cell induced and regulated? It

appears to be completely out of control in infected susceptible

BALB/c mice, but fairly under control in infected relatively resis-

tant C57BL/6 mice (6). Do Tp cells, generated in the spleen, in-

vade the liver or any other infected organs, such as the brain in

Trypanosoma brucei rhodesiense infections?

In summary, we conclude that MHC-II-restricted CD4ϩ Tp cells

exert mortality via secretion of IFN-␥ in susceptible mice infected

with a virulent strain of T. congolense.

AcknowledgmentsWe thank Dr. Tak Mak for providing two breeding pairs of CD8- and

CD4-deficient BALB/c mice. We are grateful to Brian Chelack (Prairie

Diagnostic Services, Saskatoon, Canada) for his assistance with the FACS

analyses. We also thank Juliane Deubner (Western College of Veterinary

Medicine) for drawing the diagram of the synopsis.

DisclosuresThe authors have no financial conflict of interest.

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Discussion.


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